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The 70 kDa heat shock protein (Hsp70) chaperones control protein homeostasis in all ATP-containing cellular compartments. J-domain proteins (JDPs) coevolved with Hsp70s to trigger ATP hydrolysis and catalytically upload various substrate polypeptides in need to be structurally modified by the chaperone. Here, we measured the protein disaggregation and refolding activities of the main yeast cytosolic Hsp70, Ssa1, in the presence of its most abundant JDPs, Sis1 and Ydj1, and two swap mutants, in which the J-domains have been interchanged. The observed differences by which the four constructs differently cooperate with Ssa1 and cooperate with each other, as well as their observed intrinsic ability to bind misfolded substrates and trigger Ssa1's ATPase, indicate the presence of yet uncharacterized intramolecular dynamic interactions between the J-domains and the remaining C-terminal segments of these proteins. Taken together, the data suggest an autoregulatory role to these intramolecular interactions within both type A and B JDPs, which might have evolved to reduce energy-costly ATPase cycles by the Ssa1-4 chaperones that are the most abundant Hsp70s in the yeast cytosol., Competing Interests: Declarations of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Leaf-feeding insects trigger high-amplitude, defense-inducing electrical signals called slow wave potentials (SWPs). These signals are thought to be triggered by the long-distance transport of low molecular mass elicitors termed Ricca's factors. We sought mediators of leaf-to-leaf electrical signaling in Arabidopsis thaliana and identified them as β-THIOGLUCOSIDE GLUCOHYDROLASE 1 and 2 (TGG1 and TGG2). SWP propagation from insect feeding sites was strongly attenuated in tgg1 tgg2 mutants and wound-response cytosolic Ca 2+ increases were reduced in these plants. Recombinant TGG1 fed into the xylem elicited wild-type-like membrane depolarization and Ca 2+ transients. Moreover, TGGs catalyze the deglucosidation of glucosinolates. Metabolite profiling revealed rapid wound-induced breakdown of aliphatic glucosinolates in primary veins. Using in vivo chemical trapping, we found evidence for roles of short-lived aglycone intermediates generated by glucosinolate hydrolysis in SWP membrane depolarization. Our findings reveal a mechanism whereby organ-to-organ protein transport plays a major role in electrical signaling., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Detailed understanding of the mechanism by which Hsp70 chaperones protect cells against protein aggregation is hampered by the lack of a comprehensive characterization of the aggregates, which are typically heterogeneous. Here we designed a reporter chaperone substrate, MLucV, composed of a stress-labile luciferase flanked by stress-resistant fluorescent domains, which upon denaturation formed a discrete population of small aggregates. Combining Förster resonance energy transfer and enzymatic activity measurements provided unprecedented details on the aggregated, unfolded, Hsp70-bound and native MLucV conformations. The Hsp70 mechanism first involved ATP-fueled disaggregation and unfolding of the stable pre-aggregated substrate, which stretched MLucV beyond simply unfolded conformations, followed by native refolding. The ATP-fueled unfolding and refolding action of Hsp70 on MLucV aggregates could accumulate native MLucV species under elevated denaturing temperatures highly adverse to the native state. These results unambiguously exclude binding and preventing of aggregation from the non-equilibrium mechanism by which Hsp70 converts stable aggregates into metastable native proteins., (© 2022. The Author(s).)

Partially charged chiral molecules act as spin filters, with preference for electron transport toward one type of spin ("up" or "down"), depending on their handedness. This effect is named the chiral induced spin selectivity (CISS) effect. A consequence of this phenomenon is spin polarization concomitant with electric polarization in chiral molecules. These findings were shown by adsorbing chiral molecules on magnetic surfaces and investigating the spin-exchange interaction between the surface and the chiral molecule. This field of study was developed using artificial chiral molecules. Here we used such magnetic surfaces to explore the importance of the intrinsic chiral properties of proteins in determining their stability. First, proteins were adsorbed on paramagnetic and ferromagnetic nanoparticles in a solution, and subsequently urea was gradually added to induce unfolding. The structural stability of proteins was assessed using two methods: bioluminescence measurements used to monitor the activity of the Luciferase enzyme, and fast spectroscopy detecting the distance between two chromophores implanted at the termini of a Barnase core. We found that interactions with magnetic materials altered the structural and functional resilience of the natively folded proteins, affecting their behavior under varying mild denaturing conditions. Minor structural disturbances at low urea concentrations were impeded in association with paramagnetic nanoparticles, whereas at higher urea concentrations, major structural deformation was hindered in association with ferromagnetic nanoparticles. These effects were attributed to spin exchange interactions due to differences in the magnetic imprinting properties of each type of nanoparticle. Additional measurements of proteins on macroscopic magnetic surfaces support this conclusion. The results imply a link between internal spin exchange interactions in a folded protein and its structural and functional integrity on magnetic surfaces. Together with the accumulating knowledge on CISS, our findings suggest that chirality and spin exchange interactions should be considered as additional factors governing protein structures.

The GroEL/ES chaperonin cavity surface charge properties, especially the negative charges, play an important role in its capacity to assist intracavity protein folding. Remarkably, the larger fraction of GroEL/ES negative charges are not conserved among different bacterial species, resulting in a large variation in negative-charge density in the GroEL/ES cavity across prokaryotes. Intriguingly, eukaryotic GroEL/ES homologs have the lowest negative-charge density in the chaperonin cavity. This prompted us to investigate if GroEL’s chaperoning mechanism changed during evolution. Using a model in vivo GroEL/ES substrate, we show that the ability of GroEL/ES to buffer entropic traps in the folding pathway of its substrate was partially dependent upon the negative-charge density inside its cavity. While this activity of GroEL/ES was found to be essential for Escherichia coli, it has been perfected in some organisms and diminished in others. However, irrespective of their charges, all the tested homologs retained their ability to regulate polypeptide chain collapse and remove enthalpic traps from folding pathways. The ability of these GroEL/ES homologs to buffer mutational variations in a model substrate correlated with their negative-charge density. Thus, Hsp60/10 chaperonins in different organisms may have changed to accommodate a different spectrum of mutations on their substrates.

Life is a non-equilibrium phenomenon. Owing to their high free energy content, the macromolecules of life tend to spontaneously react with ambient oxygen and water and turn into more stable inorganic molecules. A similar thermodynamic picture applies to the complex shapes of proteins: While a polypeptide is emerging unfolded from the ribosome, it may spontaneously acquire secondary structures and collapse into its functional native conformation. The spontaneity of this process is evidence that the free energy of the unstructured state is higher than that of the structured native state. Yet, under stress or because of mutations, complex polypeptides may fail to reach their native conformation and form instead thermodynamically stable aggregates devoid of biological activity. Cells have evolved molecular chaperones to actively counteract the misfolding of stress-labile proteins dictated by equilibrium thermodynamics. HSP60, HSP70 and HSP100 can inject energy from ATP hydrolysis into the forceful unfolding of stable misfolded structures in proteins and convert them into unstable intermediates that can collapse into the native state, even under conditions inauspicious for that state. Aggregates and misfolded proteins may also be forcefully unfolded and degraded by chaperone-gated endo-cellular proteases, and in eukaryotes also by chaperone-mediated autophagy, paving the way for their replacement by new, unaltered functional proteins. The greater energy cost of degrading and replacing a polypeptide, with respect to the cost of its chaperone-mediated repair represents a thermodynamic dilemma: some easily repairable proteins are better to be processed by chaperones, while it can be wasteful to uselessly try recover overly compromised molecules, which should instead be degraded and replaced. Evolution has solved this conundrum by creating a host of unfolding chaperones and degradation machines and by tuning their cellular amounts and activity rates., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fauvet, Rebeaud, Tiwari, De Los Rios and Goloubinoff.)

Understanding the folding pathway of any protein is of utmost importance for deciphering the folding problems under adverse conditions. We can obtain important information about the folding pathway by monitoring the folding of any protein from its unfolded state. It is usually very difficult to monitor the folding process in real time as the process is generally very fast, and we need a suitable read out. In this protocol, we have solved this issue by using a protein that is non-fluorescent in its unfolded state but fluoresces in its native state after folding. The kinetics of refolding can be monitored by following the increase in fluorescence in real time. Previously, this was generally achieved by either monitoring a protein's enzymatic activity or measuring the tryptophan fluorescence, where the signal output depends on well-described enzymatic activity or the frequency of tryptophan residues present in the proteins, respectively. Here, we describe a simple and real-time assay to monitor the refolding of sGFP, a recently described slow-folding mutant of yeGFP (yeast enhanced GFP). We unfold this protein using chemical denaturant and refold in a suitable buffer, monitoring the increase in fluorescence over time. GFP is fluorescent only when correctly folded; thus, using this technique, we can measure the true rate of protein refolding by following the increase in fluorescence over time. Therefore, sGFP can be used as an ideal model to study the in vitro protein folding process. Accordingly, the effects of different conditions and molecules on the protein folding pathway can be efficiently studied using sGFP as a model protein. Graphical abstract: Schematic of the steps involved in the sGFP refolding pathway. Native sGFP is unfolded by chemical denaturation using 6 M GuHCl at 25°C for 1 hour and then refolded in refolding buffer by 100-fold dilution., Competing Interests: Competing interestsThe authors declare no competing interests., (Copyright © 2021 The Authors; exclusive licensee Bio-protocol LLC.)

The folding landscape of proteins can change during evolution with the accumulation of mutations that may introduce entropic or enthalpic barriers in the protein folding pathway, making it a possible substrate of molecular chaperones in vivo. Can the nature of such physical barriers of folding dictate the feasibility of chaperone-assistance? To address this, we have simulated the evolutionary step to chaperone-dependence keeping GroEL/ES as the target chaperone and GFP as a model protein in an unbiased screen. We find that the mutation conferring GroEL/ES dependence in vivo and in vitro encode an entropic trap in the folding pathway rescued by the chaperonin. Additionally, GroEL/ES can edit the formation of non-native contacts similar to DnaK/J/E machinery. However, this capability is not utilized by the substrates in vivo. As a consequence, GroEL/ES caters to buffer mutations that predominantly cause entropic traps, despite possessing the capacity to edit both enthalpic and entropic traps in the folding pathway of the substrate protein., Competing Interests: Declarations of Interest The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Mortality in COVID-19 patients predominantly results from an acute respiratory distress syndrome (ARDS), in which lungs alveolar cells undergo programmed cell death. Mortality in a sepsis-induced ARDS rat model is reduced by adenovirus over-expression of the HSP70 chaperone. A natural rise of body temperature during mild fever can naturally accumulate high cellular levels of HSP70 that can arrest apoptosis and protect alveolar lung cells from inflammatory damages. However, beyond 1-2 h of fever, no HSP70 is being further produced and a decreased in body temperature required to the restore cell's ability to produce more HSP70 in a subsequent fever cycle. We suggest that antipyretics may be beneficial in COVID-19 patients subsequent to several hours of mild (<38.8°C) advantageous fever, allowing lung cells to accumulate protective HSP70 against damages from the inflammatory response to the virus SARS-CoV-2. With age, the ability to develop fever and accumulate HSP70 decreases. This could be ameliorated, when advisable to do so, by thermotherapies and/or physical training., (Copyright © 2020 Guihur, Rebeaud, Fauvet, Tiwari, Weiss and Goloubinoff.)

In eukaryotes, Hsp110s are unambiguous cognates of the Hsp70 chaperones, in primary sequence, domain organization, and structure. Hsp110s function as nucleotide exchange factors (NEFs) for the Hsp70s although their apparent loss of Hsp70-like chaperone activity, nature of interdomain communication, and breadth of domain functions are still puzzling. Here, by combining single-molecule FRET, small angle X-ray scattering measurements (SAXS), and MD simulation, we show that yeast Hsp110, Sse1 lacks canonical Hsp70-like interdomain allostery. However, the protein exhibits unique noncanonical conformational changes within its domains. Sse1 maintains an open-lid substrate-binding domain (SBD) in close contact with its nucleotide-binding domain (NBD), irrespective of its ATP hydrolysis status. To further appreciate such ATP-hydrolysis-independent exhaustive interaction between two domains of Hsp110s, NBD-SBD chimera was constructed between Hsp110 (Sse1) and Hsp70 (Ssa1). In Sse1/Ssa1 chimera, we observed undocking of two domains leading to complete loss of NEF activity of Sse1. Interestingly, chimeric proteins exhibited significantly enhanced ATPase rate of Sse1-NBD compared to wild-type protein, implying that intrinsic ATPase activity of the protein remains mostly repressed. Apart from repressing the high ATPase activity of its NBD, interactions between two domains confer thermal stability to Sse1 and play critical role in the (co)chaperoning function of Sse1 in Ssa1-mediated disaggregation activity. Altogether, Sse1 exhibits a unique interdomain interaction, which is essential for its NEF activity, suppression of high intrinsic ATPase activity, co-chaperoning activity in disaggregase machinery, and stability of the protein., (© 2019 Federation of European Biochemical Societies.)

Many proteins comprising of complex topologies require molecular chaperones to achieve their unique three-dimensional folded structure. The E.coli chaperone, GroEL binds with a large number of unfolded and partially folded proteins, to facilitate proper folding and prevent misfolding and aggregation. Although the major structural components of GroEL are well defined, scaffolds of the non-native substrates that determine chaperone-mediated folding have been difficult to recognize. Here we performed all-atomistic and replica-exchange molecular dynamics simulations to dissect non-native ensemble of an obligate GroEL folder, DapA. Thermodynamics analyses of unfolding simulations revealed populated intermediates with distinct structural characteristics. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native β-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spectrometry (HDX-MS). Further, we constructed network graphs to elucidate long-range intra-protein connectivity of native and intermediate topologies, demonstrating regions that serve as central "hubs". Overall, our results implicate that genomic variations (or mutations) in the distinct regions of protein structures might disrupt these topological signatures disabling chaperone-mediated folding, leading to formation of aggregates.

The role of bacterial DnaJ protein as a cochaperone of DnaK is strongly appreciated. Although DnaJ unaccompanied by DnaK can bind unfolded as well as native substrate proteins, its role as an individual chaperone remains elusive. In this study, we demonstrate that DnaJ binds a model non-native substrate with a low nanomolar dissociation constant and, more importantly, modulates the structure of its non-native state. The structural modulation achieved by DnaJ is different compared to that achieved by the DnaK-DnaJ complex. The nature of structural modulation exerted by DnaJ is suggestive of a unique unfolding activity on the non-native substrate by the chaperone. Furthermore, we demonstrate that the zinc binding motif along with the C-terminal substrate binding domain of DnaJ is necessary and sufficient for binding and the subsequent binding-induced structural alterations of the non-native substrate. We hypothesize that this hitherto unknown structural alteration of non-native states by DnaJ might be important for its chaperoning activity by removing kinetic traps of the folding intermediates.

Many proteins refold in vitro through kinetic folding intermediates that are believed to be by-products of native-state centric evolution. These intermediates are postulated to play only minor roles, if any, in vivo because they lack any information related to translation-associated vectorial folding. We demonstrate that refolding intermediate of a test protein, generated in vitro, is able to find its cognate chaperone, from the whole complement of Escherichia coli soluble chaperones. Cognate chaperone-binding uniquely alters the conformation of non-native substrate. Importantly, precise chaperone targeting of substrates are maintained as long as physiological molar ratios of chaperones remain unaltered. Using a library of different chaperone substrates, we demonstrate that kinetically trapped refolding intermediates contain sufficient structural features for precise targeting to cognate chaperones. We posit that evolution favors sequences that, in addition to coding for a functional native state, encode folding intermediates with higher affinity for cognate chaperones than noncognate ones., (Copyright © 2012 Elsevier Ltd. All rights reserved.)